This invention is in the fields of pharmacology and biochemistry. It relates to the synthesis of certain complexes of L-cysteine, N-acetyl L-cysteine, N-(2-mercapto-propionyl)glycine, L-2-oxothiazolidine-4-carboxylate and the nutritional or clinical use of these and other related individual or complexed thiol contributing, glutathione predecessors. The use of these molecules and complexes in clinical presentations of chronic glaucoma, diabetes mellitus, macular degeneration, neurodegenerative diseases and vasoconstriction are described in particular.
A. Chronic Glaucoma
The eye is maintained in a homeostatic shape by a relatively stable intraocular pressure (IOP) that varies within a reasonably narrow range so long as the intraocular production of aqueous fluid remains equal to its exit from the eye.
The optic nerve head can tolerate relatively high levels of IOP if the availability of oxygen from posterior ciliary arteries and optic nerve head arterioles remains adequate. However, if the global intraocular pressure is higher than the perfusion pressure driving oxygen through the arteriole into the surrounding tissues, decreasing amounts of oxygen will reach the optic nerve head and nerve disability will result.
Similarly if nerve head arterioles are unable to provide sufficient volumes of blood to the optic nerve, dysfunction will follow. These arteriolar deficiencies may occur because of: vasoconstriction secondary to generalized or localized microvascular dysregulation, arteriolar muscular hypertrophy (perhaps as a result of chronic spasm), atherosclerotic luminal reduction, changes in the viscosity or laminar flow patterns of the arterial blood or in either essential or iatrogenic systemic hypotension.
Glaucoma in various guises affects a large segment of the public. It is estimated that 2% to 2.5% of the population over the age of 40 has chronic open angle glaucoma (COAG). This is the most common form of glaucoma.
Because optic nerve damage occurs in patients with chronically elevated IOP, present treatments concentrate on reducing this objective finding by a variety of modalities: topical eye drops, oral medications, intravenous medications, surgical procedures, laser phototherapy, etc. All of these focus upon the reduction of pressure inside the eye and rely upon this pressure reduction to prevent optic nerve damage. For many patients this approach is effective. However, the effectiveness of each of these treatments runs from total ineffectiveness, progressive optic atrophy and eventual blindness, to an arrest of the disease, complete cessation or prevention of further optic nerve failure and preservation of vision.
Factors other than IOP levels influence the clinical outcome for many glaucoma patients. This invention is focused upon two alternatives: a) hypovascularity of the optic nerve head and loss of the vascular integrity of the optic nerve resulting in glial collapse, ganglion cell apoptosis and progressive neural atrophy with visual loss; b) hypoxia-induced free radical interference with retrograde axoplasmic flow within the optical neural axons.
Ocular Microvascular Regulation
A balanced biochemistry of nitric oxide (NO) and endothelin-1 (ET-1) mediates local optical blood flow and many facets of systemic vascular autoregulation.
NO is a highly soluble gas formed within endothelial cells by the action of the constitutive enzyme nitric oxide synthetase (cNOS). NO activates guanylate cyclase and increases guanosine-monophosphate (cGMP) within the vascular musculature. cGMP produces relaxation and dilatation of vessels. It also has more generalized smooth muscle relaxing abilities; in this regard it relaxes the contractile trabecular elements of the eye, increases aqueous outflow and reduces IOP. Levels of NO in the trabecular region of eyes of glaucoma patients are lower than in the eyes of non-glaucoma patients. Aging and atherosclerotic dysfunction of the vascular endothelium reduce its ability to produce NO because of reduced local levels of cNOS.
ET-1 is also formed within and secreted by endothelial cells. ET-1 reacts with local receptors on smooth muscle cells to produce a powerful and long-lasting vasoconstriction. ET-1 is particularly released by aged or unhealthy endothelial cells, e.g., in the presence of atherosclerosis or in the presence of local collections of endothelial leukocytes or platelets, etc. The smooth muscle contraction produced by ET-1 strongly opposes the relaxation properties of NO and trabecular contraction is stimulated, resistance to aqueous outflow is increased and IOP increases. Aqueous levels of ET-1 are elevated in glaucomatous eyes. Induced elevations of aqueous ET-1 levels produce optic nerve collapse.
This balance between NO and ET-1 mediates the autoregulation of blood flow within the optic nerve and throughout the peripheral circulation.
Exposure of patients to calcium channel blockers has resulted in an improvement of some glaucomatous visual fields. Vascular endothelial production of ET-1 is dependent upon cytosolic calcium (Ca2+) influx via transmembrane calcium channels. Calcium channel blockade reduces this Ca2+ influx and reduces the production of ET-1. A serendipitous reduction of IOP has been observed as a side effect in glaucoma patients using calcium channel blockers for systemic hypertension. However, prescribing therapeutic doses of calcium channel blockers to non-hypertensive glaucoma patients subjects the optic nerve to a risk of hypoxia secondary to iatrogenic hypotension and severely disrupts inherent transmembrane calcium modulation.
Ocular Vascular Disease
In the optic nerve two tissues are particularly vulnerable to hypoxia:
a. The microglial ganglion cells.
b. The transiting axonic neurons.
A reduction in optic nerve oxygen delivery may follow acute or chronic, segmental or widespread, vascular spasm or prolonged constriction secondary to a physical reduction in the vascular lumen. This luminal reduction (vasoconstriction) may be caused by or associated with hypertrophy of the vascular muscle wall (the media), the accumulation of atherosclerotic plaque, platelet agglutination and/or local inflammatory swelling and leukocytic accumulation. Any and all of these findings may occur with aging and with systemic disease: diabetes, hypertension, dyslipogenesis, arteriosclerosis, thyroid disease, etc. Although vascular insufficiency at specific tissue sites is widely variable and not predictable with certainty, the fact that most glaucoma patients are over 50 years old makes the frequency of these risk factors and the frequency of vascular insufficiency high in this clinical group.
If a reduction of optic nerve vascular risk factors is united with a reduction in outflow resistance, the combined effects of a more pressure resistant nerve head and lower IOP will beneficially decrease the potential for optic atrophy and blindness.
Glaucomaxe2x80x94Present Treatment
Current non-surgical treatments of COAG are based upon a limited number of biochemical approaches and focus exclusively upon reducing IOP:
a. Enzyme poisonsxe2x80x94these are most frequently tablets of carbonic anhydrase inhibitors that inhibit the production of aqueous humor. Besides the development of renal stones, potassium loss is a constant clinical concern. Topical forms of this group have appeared as eye drops. However, because carbonic anhydrase activity is also present in the cytoplasm of corneal endothelial cells the long-term corneal effects of this form of these medications are unknown. To avoid systemic reactions, patients with sulfonamide allergies should not use these drugs.
b. Parasympathomimeticsxe2x80x94pilocarpine-containing eye drops are widely prescribed and act by causing pupillary constriction. Miosis causes lacunae in the trabeculum to enlarge; thus, mechanical resistance to aqueous outflow is reduced. Frequent side effects include headache from iris spasm, decreased night vision from miosis and blurred vision, especially in myopes.
c. Beta blocking agentsxe2x80x94these drugs block the beta-adrenergic sympathetic rete responsible for increased vascular flow to the ciliary processes and reduce the production of aqueous humor. They also increase aqueous outflow through the trabeculum. These agents must be used with great caution in patients with low blood pressure (orthostatic hypotension), sinus bradycardia or second/third degree heart block (severe bradycardia), obstructive pulmonary disease or bronchial asthma (acute bronchospasm) and diabetes (masking of hypoglycemia). They result in impotency in a significant number of men. There is contested evidence that some ocular beta blocking agents generally reduce blood flow to the posterior segment of the eye.
d. Topical prostaglandin analogsxe2x80x94this new group of anti-inflammatory eye drops presumably reduces IOP by widening the inter-trabecular space and, perhaps, by reducing trabecular platelet aggregation. Their use is associated with progressive and possibly permanent change in iris color to brown and some embryocidal outcomes in laboratory animals. Women of reproductive age and nursing women should avoid their use.
All of these treatment modes have significant and unavoidable, potential or demonstrable, local or systemic side effects or toxicities that directly contraindicate their use, reduce patient compliance or are worrisomely interactive with other systemic pharmaceuticals.
B. Non-Insulin-Dependent Diabetes Mellitus (NIDDM)
Non-insulin-dependent diabetes is prevalent in up to 35% of the population. It is most frequently a disorder of middle and later life. It is both part of the aging process and a process that advances aging. Diabetes affects metabolism in totality: carbohydrate, lipid and protein.
There are two clinical forms of diabetes, each with a different pathogenesis: Type 1, insulin-dependent diabetes mellitus (IDDM) and Type 2, non-insulin-dependent diabetes mellitus (NIDDM). NIDDM represents 90% of all diabetics. In NIDDM, cellular resistance to the effectiveness of insulin results in above normal levels of insulin secretion. When this compensatory increase of insulin production cannot be maintained and/or when insulin resistance increases further, blood sugar rises, lipid and protein metabolism are disturbed and the insidious processes of vascular complications of long-term diabetes begin.
Diabetes is characterized by a congeries of pathologies other than hyperglycemia; most seriously, patients develop specific microvascular and non-specific macrovascular complications including retinopathy, nephropathy, neuropathy and frequently severe atherosclerosis affecting, among others, the coronary, cerebral and peripheral vascular trees. Causative mechanisms of these complications include free radical damage, non-enzymatic protein glycation, lipoprotein disturbances and disorders of sorbitol and myoinositol metabolism.
Insulin resistance with secondary hyperinsulinemia and/or hyperglycemia disturbs several physiological conditions and functions and, thus, contributes to many disorders associated with aging, e.g., hypertension, obesity, atherosclerosis, lipid abnormalities and chronic metabolic perturbations including fully developed NIDDM.
In diabetes, as in aging, elevated circulating glucose reacts non-enzymatically with proteins and nucleic acids to form products that: 1) disturb the functionality of the cellular phospholipid membrane, 2) diminish tissue elasticity and 3) increase lipid peroxidation.
Disturbances in glucose/insulin metabolism are associated with greatly increased lipid peroxidation from elevated free radical formation resulting from the auto-oxidation of glucose. This augmented free radical formation and lipid peroxidation are associated with the xe2x80x9cpremature agingxe2x80x9d of diabetic patients.
Ingestion of sugars, fats and sodium have been linked to decreased insulin sensitivity, while caloric restriction, exercise, ingestion of chromium, vanadium, magnesium, and certain antioxidants have been associated with greater insulin sensitivity. Thus, manipulation of the diet by influencing the glucose/insulin system may favorably affect lifespan and reduce the incidence of the microvascular and macrovascular complications of NIDDM.
The earliest microvascular lesion of diabetes is thickening of the basement membrane. A healthy basement membrane provides stability and a permeability barrier. Cellular impermeability requires a negative electrical charge provided by heparan sulfate, a proteoglycan. Sulfate groups provided by thiol contributors like xcex1-lipoic acid and N-acetylcysteine (NAC) may contribute to the adequacy of this necessary negativity of the cell membrane. In diabetes both the basement membrane thickness and heparan sulfate levels are decreased, as is overall membrane sulphonation. As a result, vessel permeability is increased. Increased vessel permeability is the most notable initial microvascular complication in diabetes.
Although arteriolar and capillary microvascular intraluminal pressure and flow may be increased, laminar flow is disordered by clumping of cellular elements. These disturbances, plus the increased permeability of the basement membrane and associated vascular endothelial dysfunction, limit normally efficient vascular autoregulatory mechanisms, and the latter eventually leads to clinically apparent microvascular and macrovascular insufficiencies of the legs, feet, heart, eye and brain.
NIDDMxe2x80x94Present Treatment
Current pharmacological approaches focus upon improving glucose homeostasis, but frequently do not succeed in permanently restoring normoglycemia in most patients.
For glycemic regulation, four classes of drugs are currently available: sulphonylureas, biguanides, alpha-glucosidase inhibitors and insulin. Adjunct treatments may help to improve glycemic control by correcting selected abnormalities associated with NIDDM, such as obesity and hyperlipidemia.
C. Vasoconstriction
Vasoconstriction, or a reduction in the cross-sectional area of the lumen of blood vessels, is due either to vasospasm, inadequate vasodilatation, thickening of the vessel wall, or the accumulation of flow-restricting materials on the internal wall surfaces or within the wall itself. Vasoconstriction is a major presumptive or proven factor in aging and in various clinical conditions including progressive generalized atherogenesis, myocardial infarction, stroke, hypertension, glaucoma, macular degeneration, migraine, hypertension and diabetes mellitus among others.
Vasoconstriction originates in a variety of ways. One example is the local conversion of circulating low density lipoproteins (LDL) into oxidatively activated low density lipoproteins (oxLDL), which are internalized via cellular macrophage scavenger receptors called xe2x80x9cfoam cellsxe2x80x9d. These cells are bound to the vascular endothelium, release cytokines and trigger local expression of leukocyte adhesion molecules.
Another example is the unopposed endothelial cell release of the vasoconstrictor, ET-1. Prolonged vasospasm results in proliferation of vascular smooth muscle cells (VSMC) and a mechanical reduction of luminal cross-section. In particular, oxLDL and hyperlipidemia impair endothelial-dependent vascular relaxation because of the inhibition of histamine-stimulated release of NO from endothelial cells. This induces a sometimes-inadequate adaptive increase in the level of intracellular glutathione (GSH) in VSMC.
A third example is the free radical-stimulated activation, local accumulation, and adhesion of platelets and white blood cells on the endothelial surface which produce chemoattractants for macrophages that eventually will be converted into xe2x80x9cfoam cellsxe2x80x9d.
A fourth example is the irregular vasoconstriction or vascular aneurismal pouching due to the death of perivascular pericytes caused by the conversion of glucose to sorbitol in diabetes mellitus.
Vasoconstriction and atherogenesis can be modulated by a number of mechanisms: inhibition of LDL oxidation by xcex1-tocopherol (vitamin E) and ascorbate (vitamin C); limitation of the production of ROS and, thus, cell-mediated LDL oxidation; reduction of adhesion molecule expression and monocyte recruitment; protection for the release of NO and reduction in the proliferation of VSMC, etc. Many, if not most, of these processes are regulated by nuclear factor-kappa B or related transcription factors that are redox-sensitive and capable of modification by antioxidants. Furthermore, antioxidants directly limit the cytotoxic effects of oxLDL and thereby reduce vascular cell necrosis and lesion progression.
The main oxidizing free radicals are oxygen-derived metabolites, such as: superoxide anion (O.), hydrogen peroxide (H2O2), hydroxyl radical (OHxe2x88x92), hypochlorous acid (HOCl), chloramines (NH2Cl), nitrogen oxides (NO.), ozone (O3) and lipid peroxides. They are produced continually by living organisms, either in the intracellular compartment by the mitochondrial respiratory chain and mixed function oxidase system, or in the extracellular compartment, especially by phagocytes. The body possesses complex protective antioxidant systems against this potentially toxic environment. These systems include dismutase superoxides, catalases, metallic ion sequestration, enzymes which degrade proteins damaged by free radicals, metabolizing hydroperoxides, inherent DNA repair processes, and in particular, the GSH enzyme system. A physiological steady state is established during healthy, normal conditions between the production of oxidants and their neutralization by antioxidants.
A. Glutathione
Human GSH (gamma-glutamyl-cysteinyl-glycine) levels cannot be raised directly by supplemental administration in the diet. GSH is produced inside the cell from the amino acids glutamic acid, cysteine and glycine and acts as a cofactor for protective enzymes such as selenium-dependent glutathione peroxidase (GSHPx). Zinc is a necessary trace element in its synthesis. GSH presence in the brain is enhanced by pineal melatonin via this neurohormone""s ability to increase the mRNA of GSHPx.
Reduced GSH is important and ubiquitous. It is necessary for intracellular transduction signaling, for the modulation of cellular apoptosis and necrosis, and the modulation of red blood cell fragility. During its function as an antioxidant it is oxidized to disulfide glutathione (GSSG). This action importantly protects vascular endothelium from free radical damage. GSH inhibits the peroxidation of LDL directly reducing atherosclerotic and vasoconstrictive risks, and oxLDL-induced mitochondrial DNA mutations. Besides their influence upon atherogenesis and vasoconstriction, these effects are linked to a variety of specific sensory neuropathies.
GSH and Neurodegenerative Diseases
GSH plays multiple roles in the nervous system including free radical scavenging, redox modulation of ionotropic receptor activity and neurotransmission. GSH depletion enhances oxidative stress and increases the level of neuroexcitotoxic molecules; in distinct neuronal populations both of these events can initiate cell death. Evidence for the dual roles of oxidative stress and diminished neural GSH status is present in Lou Gehrig""s disease (ALS), Parkinson""s disease and Alzheimer""s disease.
Exposure to glutamate, a critical neurotransmitter, causes depletion of intracellular mitochondrial GSH leading to the accumulation of ROS and, ultimately, neural apoptosis. Cells that have enhanced rates of GSH regenerationxe2x80x94due to higher activities of the GSH metabolic enzymes gamma-glutamylcysteine synthetase and GSH reductasexe2x80x94appear to be resistant to glutamate-induced ROS.
Neurodegenerative disorders occurring with age, e.g. Alzheimer""s disease and prion-based diseases like Creutzfeldt-Jakob disease are associated with a reduction of GSH levels. Normalization of the GSH level appears to exert a neuroprotective effect.
(Also see GSH relationship with CNS metallothioneins, below)
GSH and Aging
GSHPx levels appear to rise with aging; this may reflect a physiological attempt to provide compensatory increases in the GSH needed to counter the rising levels of ROS associated with increasing age.
Because the protection of the electron acceptor homocysteine thiolactone declines with aging, homocysteine levels frequently increase. GSH levels are lowered by homocysteine.
GSH is low in the presence of hypomagnesemia. Hypomagnesemia is commonly present in the aging (and the diabetic) population.
GSH increases the oxidative stability of muscle tissue and presumably improves aging muscular tone.
GSH and Diabetes
The elevated oxidative stresses associated with hyperglycemia may be involved in the abnormal activation of the coagulation cascade found in diabetics. Prothrombin fragment 1+2 (F1+2) represents a reliable marker of the amount of thrombin released. During oral glucose tolerance tests, F1+2 significantly increases in both diabetic and healthy subjects. Intravenous GSH administration during these tests normalizes this phenomenon and significantly decreases F1+2 in diabetics.
Reduced GSH is a cofactor for the glyoxalase system, a metabolic pathway that catalyses the detoxification of xcex1-oxoaldehydes (RCOCHO) to corresponding aldonic acids (RCH(OH)CO2H). This detoxification protects cells from xcex1-oxoaldehyde-mediated formation of advanced glycation endproducts (AGEs). AGEs are implicated in a wide variety of diabetic vascular abnormalities and, perhaps, in the pathogenesis of macular degeneration.
Polyol-(sorbitol) induced decreases in nicotinamide adenine dinucleotide phosphate (NADPH) in erythrocytes from patients with NIDDM impair the redox status of GSH. Since activation of the polyol pathway is significant in diabetes, decreases in NADPH and GSH levels occur.
Retinal gamma-glutamyl transpeptidase (GTT) activity and GSH levels are significantly reduced in diabetic and galactosemic rats. Consumption of the antioxidants ascorbic acid plus xcex1-tocopherol inhibits these decreases of retinal GTT activity and GSH levels. This suggests that defects in GSH regulation in the diabetic retina are secondary to hyperglycemia-induced oxidative stress.
A significantly lower content of sulfhydryl proteins is present in the lens and vitreous of diabetic patients. This is associated with an increased formation of protein-bound free sulfhydryls, one index of oxidative damage to proteins. In addition, GSHPx activity is decreased in the lenses of diabetic patients. Presumably this would result in reduced levels of GSH in the diabetic lens.
Free radicals have been proposed as fundamental to the development of diabetic retinopathy because they are routinely produced in high volume by the abnormal metabolism of diabetes. Microvascular ischemia/reperfusion cycles, which interfere with the FR enzyme defense system of the retina, i.e., with GSH, are also implicated.
GSH and the Eye
The ciliary body in particular appears to contain an inducible and very active mono-oxygenase system prone to ROS generation. These ROS, combined with those produced via the cyclo-oxygenase pathway probably result in damage through oxidative stress-mediated vascular constriction.
In the retina the photoreceptor rhodopsin itself may be the photodynamic agent that initiates ROS formation. High concentrations of retinal polyunsaturated fatty acids (PUFAs) in the photoreceptor membranes form additional ROS by auto-oxidation.
Fatty acids, e.g., C22:6 omega 3, are especially concentrated in rods and cones and in the phosphatidyl ethanolamine of retinal synaptosomes. As a result of peroxidation, malondialdehyde is formed. This aldehyde appears to cross-link the amino groups of proteins with phospholipids, which results in the production of retinal lipofuscin. From this source drusen are formed. The latter are precursors of senile macular degenerationxe2x80x94a major source of visual disability in the aging population.
The protective antioxidative capacity of the youthful and healthy ciliary body is correspondingly very high (especially via SOD and GSH). Toxic peroxidation processes in particular are countered by these enzyme systems and antioxidants. However, rapid oxidation of ascorbate in the aqueous yields H2O2, which itself is locally toxic to endothelial cells. A potentially important, relationship may exist between unmodulated aqueous increases of H2O2 and H2O2-derived toxic ROS (e.g., OH+), and the development of various ocular pathologies such as glaucoma, cataract, macular degeneration and retinal vascular damage, including the neovascularization of prematurity. This oxidation of ascorbate in the aqueous humor is limited by GSH.
GSH and Vasoconstriction
Redox-sensitive mechanisms are involved in VSMC growth. ROS that promote VSMC growth are inhibited by GSH. This is not surprising since, upon oxidation, micronutrients need to be regenerated in the biological setting, hence their need for coupling to complex, often redundant, nonradical-reducing systems such as GSH/GSSH or NADPHINADP+ and NADHINAD+. For example: the water-soluble, antioxidant vitamin C can reduce oxidized vitamin E tocopheroxyl radicals directly or indirectly; however, other reducing compounds such as xcex1-lipoic acid and GSH can also perform these functions.
An inverse correlation exists between the extent of macrophage-mediated oxidation of LDL and cellular GSH content. Supplemental thiols which increase GSH levels should protect endothelial cells from atherosclerotic damage, perturbations of laminar flow, VSMC hypertrophy, cell detachment, et al, and thus help to preserve a normal NO/ET-1 ratio.
However, some details of the protective functions of GSH function remain unclear. Electrophoretic mobility shift assays demonstrate that activation of oxLDL and tumor necrosis factor alpha (TNF alpha) is not attenuated by GSH or by cGMP analogues.
B. GSH Thiol Contributors
a. Cysteine
Cysteine is a necessary thiol precursor of GSH. Cysteine is a powerful scavenger of peroxynitrite, an extremely toxic free radical that is responsible for DNA damage, decreases in mitochondrial respiration and the loss of cellular levels of NAD+ [69]. Additionally cysteine reduces arachidonic acid release, prostaglandin E2 synthesis and lipid peroxidation, all events associated with inflammatory states.
b. N-acetyl L-cysteine (NAC)
As mentioned above, oxLDL induces apoptosis in human macrophages, a significant feature of atherogenesis. However, cell cultures exposed to NAC before they are exposed to oxLDL, TNF-alpha or H2O2, do not experience decreases in cellular GSH concentrations. This is especially true in apoptotic macrophages present in human atherosclerotic plaques. NAC has a GSH sparing effect under these circumstances.
In another supporting study, NAC inhibited inflammatory interleukin (IL-8) expression induced by TNF-alpha. Such local inflammatory elements are increasingly implicated in vascular atherosclerotic changes associated with cardiac disease.
c. L-2-oxothiazolidine-4-carboxylate (OTC)
Cellular oxygenases and antioxidants, including GSH, modulate macrophage-mediated oxidation of LDL in early atherogenesis. OTC delivers cysteine residues to the cells for GSH synthesis. Supplementation with OTC (and selenium which increases cellular GSH synthesis) seems to increase macrophage GSH content and GSHPx activity. OTC should reduce cellular oxLDL production.
Increased vascular oxidative stress impairs the effective vasorelaxation action of NO in atherosclerosis. NO action is improved by the administration of ascorbic acid (which regulates intracellular redox states) perhaps by sparing cellular GSH. By providing substrate cysteine for GSH synthesis and thus augmenting intracellular GSH, OTC improves NO-dependent, flow-mediated dilatation. At the same time OTC has no effect on direct arterial dilation caused by nitroglycerin or upon systemic blood pressure, heart rate, or reactive hyperemia.
d. N-(2-Mercaptopropionyl)glycine (MPG)
MPG is a reducing, radical scavenging, antioxidant agent that decreases hydroxyl concentration and the hypoxic induction of mRNA. Other studies have shown that MPG also prevents the reduction of tyrosine hydroxylase mRNA by H2O2.
Antioxidants are known to mitigate the cardiac contractile dysfunction that follows brief periods of ischemia (xe2x80x9cmyocardial stunningxe2x80x9d); following such ischemia, both re-flow and isovolumic pressures recovered completely in a MPG treated group.
Studies have been made of the presumably antioxidant radioprotective effects of MPG upon the cells of bone marrow in irradiated mice. MPG pre-treatment of the mice resulted in a significant reduction in the percentage of aberrant metaphases.
C. Magnesium (Mg+2)
Although the recommended daily allowance of ionic Mg+2 for humans is 350 mg. Mg+2 deficiencies have been documented in many segments of the world population. The average adult in Western society has a dietary Mg+2 shortfall of 90-178 mg. per day. Mg+2 deficiencies are particularly prevalent among diabetics with normal renal function, alcoholics, smokers, the elderly, and those who suffer from a variety of gastrointestinal mobility disorders.
Ionic Mg+2 in mammals resides in three compartments: (1) in bone; (2) in an intracellular bound form or in an intracellular unbound form; and (3) in circulating bound and unbound forms. When the concentration of circulating Mg+2 in the bloodstream increases as a result of dietary uptake of Mg+2, the body responds by attempting to sequester the Mg+2 into one of the bound or intracellular forms listed above. However, if elemental Mg+2 is rapidly ingested in a bulk amount that results in the absorption of a Mg+2 bolus in excess of 8 mEq, the renal excretion of Mg+2 quickly increases and becomes less efficient in the resorption of this element. Thus the accurate sustenance of an appropriate Mg+2 level requires the repeated administration of carefully designed medicaments with correctly formulated, targeted amounts.
Mg+2 deficiencies impair antioxidant defenses through decreased synthesis of GSH and a reduced activity of CuZnSOD. Mg+2 deficiencies enhance general oxidative stress levels by raising circulating levels of factors that promote free radical generation and which are mitogenic. This may result in increased tissue necrosis in the presence of acute local levels of active oxygen species or hydroxyl radicals.
D. Copper (Cu+2)
Cu+2 is an essential trace element required for a number of enzymes that are necessary for normal metabolic function. Metabolic balance studies have demonstrated that daily Cu+2 losses are approximately 1.3 mg/day. In order to remain in Cu+2 balance, the average adult male must consume a diet that contains at least 2 mg copper/day. It has been assumed that most diets satisfy this requirement because of the ubiquitous presence of Cu+2 in most foodstuffs. Recent studies, however, have shown that dietary Cu+2 may often fall below the estimated daily needs.
The essential yet toxic nature of Cu+2 demands tight regulation of the Cu+2 homeostatic machinery to ensure that sufficient Cu+2 is present in the cell to drive essential biochemical processes yet prevent accumulation to toxic levels.
The results of some studies demonstrate that Cu+2 deficiency results in alterations of the regulatory mechanisms governing inflammation and thrombosis.
Cu+2 is strongly involved in the synthesis of GSH and is necessary for the activity of the antioxidant CuZnSOD.
E. Zinc (Zn+2)
Compared with controls, rats fed a Zn+2-deficient diet without supplementary antioxidants have greater red blood cell osmotic fragility, higher concentrations of thiobarbituric acid-reactive substances (TBARS), higher GSHS-transferase activity, lower concentration of GSH and of GSHPx, as well as lower activity of CuZnSOD. High dietary levels of Zn+2 appear to reduce levels of CuZnSOD. In one study there was no relationship between serum Zn+2 levels and CuZnSOD activity or the serum concentration of GSHPx activity in a group of healthy subjects. However, in elderly subjects given Zn+2 supplements for one year, mean plasma levels of xcex1-tocopherol, vitamin C and Cu+2 increased significantly after 6 months of supplementation. A significant increase in GSHPx levels was observed in patients receiving these trace elements alone or in association with vitamins.
Zn+2 binds the sulfhydryl groups in proteins, protecting them from oxidation. Zn+2 status does not directly control tissue peroxide levels but can protect specific molecules against oxidative and peroxidative damage.
Many areas of the brain contain high contents of Zn+2: the retina, the pineal gland (note relationship to the pineal antioxidant, melatonin) and the hippocampus all synthesize unique metallothioneins (MT) on a continuous basis. MT are Zn+2-binding proteins consisting of 25-30% cysteine. GSH may participate in releasing Zn+2 from MT. The concentration of Zn+2 is altered in a number of disorders of the central nervous system: alcoholism, Alzheimer""s dementia, Down syndrome, epilepsy, Friedreich""s ataxia, Guillaine-Barrxc3xa9 syndrome, hepatic encephalopathy, multiple sclerosis, Parkinson""s disease, Pick""s disease, retinitis pigmentosa, retinal dystrophy, schizophrenia, and Wemicke-Korsakoff syndrome.
The invention resides in the synthesis and application of unique, efficient molecules presented in dosage forms clinically useful as nutritional supplements for, among others, chronic glaucoma, diabetes, macular degeneration, neurodegenerative diseases and vasoconstriction. It introduces a variety of molecules unique in design and/or in application.
1. Metal thiol complexes included in this invention have the following formula
[A]M X
wherein
a. A is L-cysteine, NAC, OTC or MPG,
b. M is Mg+2, Cu+2, Zn+2 or Se+2,
c. X is hydroxide, halide, acetate or ascorbate, prepared in oral unit dosage forms clinically useful for chronic glaucoma, NIDDM, macular degeneration, neurodegenerative diseases or vasoconstriction, among others.
2. Additional metal thiol complexes included in this invention have the following formula
[A]2MX
wherein
a. [A]2 is bis-L-cysteine, bis-NAC, bis-OTC or bis-MPG,
b. M is Mg+2, Cu+2, Zn+2 or Se+2,
c. X is hydroxide, halide, acetate or ascorbate, prepared in oral unit dosage forms clinically useful for chronic glaucoma, NIDDM, macular degeneration, neurodegenerative diseases or vasoconstriction, among others.
3. Non-metal containing thiol complexes included in this invention have the following formula
[A]X
wherein
a. A is L-cysteine, NAC, OTC or MPG,
b. X is hydroxide, halide, acetate, ascorbate or bis-ascorbate, prepared in oral unit dosage forms clinically useful for the alteration of conditions and functions associated with aging, chronic glaucoma, NIDDM, macular degeneration, neurodegenerative diseases and vasoconstriction.
4. Additional non-metal containing thiol complexes included in this invention have the following formula
[A]2X
wherein
a. [A]2 is bis-L-cysteine, bis-NAC, bis-OTC or bis-MPG,
b X is hydroxide, halide, acetate, ascorbate or bis-ascorbate, prepared in oral unit dosage forms clinically useful for the alteration of conditions and functions associated with aging, chronic glaucoma, NIDDM, macular degeneration, neurodegenerative diseases and vasoconstriction.
Processes for the Synthesis of Salts:
The magnesium and zinc salts of N-acetyl-L-cysteine and L-cysteine are prepared as described in U.S. Pat. Nos. 3,647,834 and 3,749,770 by treating 2 molar equiv. of the carboxylic acids with one molar equiv. of the carbonate salts of magnesium and zinc respectively. The magnesium and zinc salts of L-2-oxothiazolidine-4-carboxyoic acid and N-(2-mercaptopropionyl)glycine are prepared in a similar manner. Alternatively, the magnesium salts are prepared by treating 2 molar equiv. of the carboxylic acid with 1 molar equiv. of magnesium ethoxide. In the foregoing preparations, replacement of one molar equiv. of acid with one molar equiv. of ascorbic acid gives the salt of the acid and ascorbic acid in a 1:1 molar ratio. The copper salts of L-cysteine, N-acetyl-L-cysteine and L-2-oxothiazolidine-4-carboxyoic acid are prepared according to the method described in U.S. Pat. No. 4,089,969 and J. Amer. Chem. Soc. 82,4174 (1960) whereby the corresponding potassium salts (2 molar equiv.) of these acids are treated with one molar equivalent of cupric nitrate. Alternatively, the copper salts are prepared by treatment of the acid, e.g., N-(2-mercaptopropionyl)glycine, with an alcoholic solution of cupric acetate as descried in J. Chem. Soc.2545 (1957). The salts of other mixed acids, wherein the counter ion is acetate, chloride and hydroxide are also included in this invention. These salts are prepared by conventional methods that are disclosed in the Examples section below.